Methods for producing a hybrid seed product

ABSTRACT

A method for increasing production of hybrid seed of bee-pollinated crops, such as alfalfa and soybean at predetermined hybridity levels. Hybrid seed is produced using female and pollenizer plants at a selected ratio of female plants to pollenizer plants. The female plants and the pollenizer plants are intermingled in the hybrid seed production field. Prediction of percentage of hybridity at various female to pollenizer ratios allows for selection of a ratio of female plants to pollenizer plants to provide seed at a test percentage of hybridity. The percentage of hybridity may be increased post-harvest by employing techniques using seed properties such as size differential, color or density to remove a higher percentage of non-hybrid seed. The hybrid seed product is maximized at various hybridity levels. Planting according to subrows allows for separate harvesting of intermingled crops. Testing the hybrid seed product provides verification of percentage of hybridity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/409,010, filed Mar. 23, 2009, U.S. Pat. No. 8,502,019, which is acontinuation of U.S. application Ser. No. 11/146,365, filed Jun. 6,2005, abandoned, which are incorporated herein by reference in theirentireties.

BACKGROUND OF THE INVENTION

The present invention generally relates to methods for production of ahybrid seed product, and more particularly to methods for producing ahybrid seed product while increasing production of hybrid seed ofbee-pollinated crops at predetermined hybridity levels, including postproduction adjustment of percentage of hybridity and verification ofpercentage of hybridity of the hybrid seed product after harvest.

Hybrid plant varieties offer many desirable agronomic traits. Hybridplant varieties often provide higher yield than non-hybrid strains.Hybrid plant varieties may also possess higher stress tolerance thannon-hybrid varieties, enabling them to survive in less favorableenvironmental conditions. Furthermore, hybrid plant varieties providegreater efficiency in breeding improvements. Hybridization allows forcombination of desirable agronomic traits from different strains.

While hybridization provides these desirable agronomic traits, certainplant species present particular challenges to hybridization. Speciessuch as corn are easily hybridized because the male and femalereproductive organs have physical separation. Other species havereproductive parts that are less accessible and have little physicalseparation between male and female parts. These species are moredifficult to hybridize. Bee-pollinated crops, such as alfalfa andsoybeans, are examples of species that have male and female reproductiveparts that have little physical separation because of the relativelysmall size of the flower.

Hybrid seed production in these species often employs cytoplasmic malesterile plants also called female plants. Seed that is produced byfemale plants from pollinations by pollenizer plants, also called maleplants, is mostly hybrid seed. Seed produced from selfing or sibbing bythe pollenizer plants is mostly non-hybrid seed. During production ofhybrid seed, employing a higher proportion of female plants topollenizer plants in a hybrid seed production field increases theproportion of hybrid seed to non-hybrid seed. However, current methodsfor producing hybrid seed result in a significant decrease of seed yieldfor each individual plant, when increasing the proportion of femaleplants to pollenizer plants. There is therefore a need for methods forincreasing the proportion of hybrid seed to non-hybrid seed whilemaintaining high overall seed yield for a hybrid seed product.

Production of hybrid varieties is subject to federal law requirementsthat make a high percentage of hybridity very desirable. 7 CFR §201.26requires that a variety have at least seventy-five percent hybridity tobe classified as a hybrid variety. Certain plant species, particularlybee-pollinated species, such as alfalfa and soybeans, present challengesto breeding plant varieties that meet this federally mandated hybriditylevel. U.S. Pat. No. 3,570,181, herein incorporated by reference,discloses a method for producing hybrid alfalfa using cytoplasmic malesterile alfalfa plants. However, production according to this methodresults in large reduction of seed yield that makes production of hybridseed of bee-pollinated crops commercially impractical. U.S. Pat. No.4,045,912, herein incorporated by reference, discloses a method forproducing seed but does not provide for production of seed meetingfederal requirements for a hybrid variety. U.S. Pat. No. 4,045,912 isfurther not concerned with verification of percentage of hybridity atcommercial levels of production. Thus, there is a need for methods forproduction of hybrid seed that provide for high seed yield whilemaintaining hybridity levels meeting federal requirements.

Production of a certified hybrid alfalfa product requires determinationof percentage of hybridity. Determination of percentage of hybridity isoften accomplished using methods employing morphological or molecularmarkers. However, using molecular markers is expensive, takessignificant time and is often commercially impractical. In some species,morphological markers require a homozygous recessive gene on one side,and dominance gene on the other side, and are therefore difficult toemploy. Furthermore, some plant species present additional difficultiesto employing such methods. For example, tetraploid species such asalfalfa have greater complexity in their genetics and inheritance.Another difficulty is the small seed size of some species, such asalfalfa. There is therefore a need for methods for determining orverifying the percentage of hybridity of a particular hybrid that avoidthese difficulties.

Separate harvesting of female seed and pollenizer seed is useful fordetermining percentage of hybridity. Prior methods of separateharvesting for species such as alfalfa require planting female seed andpollenizer seed in separate rows for separate harvesting. However,production of hybrid seed for bee-pollinated crops, such as alfalfa andsoybeans, benefits from intermingling to allow a higher percentage ofcross-pollination to occur. Planting in separate rows deceasesproduction of hybrid seed, as cross-pollination becomes less frequentdue to the distance of the female plants from the pollenizer plants.Where multiple female rows are planted for every male row, such as shownin U.S. Pat. No. 3,570,181, cross-pollination becomes even lessfrequent. There is therefore a need for methods for separatelyharvesting female seed and pollenizer seed that allow for interminglingof female and pollenizer plants.

Production of a hybrid seed product is furthermore a long, expensive andlabor-intensive process. Prior methods of seed production do not providefor prediction of percentage of hybridity. A method for predictinghybridity levels of seed production of bee-pollinated crops wouldfacilitate production of a hybrid seed product meeting federalstandards. This would allow for faster, cost-effective and lesslabor-intensive production. There is therefore a need for methods forpredicting hybridity levels of bee-pollinated crops.

Prior methods of seed production also do not include methods forpost-production adjustment of hybridity levels of bee-pollinated crops.A method for adjusting hybridity levels of bee-pollinated crops wouldalso aid meeting hybridity standards for production of hybrid plantvarieties. Post-production adjustment of hybridity levels also wouldallow for production of hybrid plant varieties while employing breedingmethods that would not normally result in hybridity levels required bystatute. There is therefore a need for post-production methods foradjusting hybridity levels of bee-pollinated crops.

Although hybrid plant varieties are desirable for agronomic reasons,commercial production of many hybrid plant varieties has not beencommercially viable, particularly for bee-pollinated crops. The presentinvention solves these needs and other problems in the field of hybridseed production by providing, in most preferred aspects, methods forproducing a hybrid seed product that includes increasing production ofhybrid seed of bee-pollinated crops at predetermined hybridity levels,prediction of percentage of hybridity, determination of percentage ofhybridity, post-production adjustment of percentage of hybridity, andverification of percentage of hybridity after harvest.

SUMMARY OF THE INVENTION

The invention therefore provides a method for producing a hybrid seedproduct of bee-pollinated crops, such as alfalfa and soybean, atpredetermined hybridity levels. Hybrid seed is produced using female andpollenizer plants at a predetermined ratio of female plants topollenizer plants mixed together in a hybrid seed production field.Prediction of percentage of hybridity at various female to pollenizerratios allows for selection of a ratio of female plants to pollenizerplants to provide seed at a selected percentage of hybridity. Thepercentage of hybridity of the hybrid seed product may be increasedafter harvesting by employing techniques using seed properties such assize differential, color or density to remove a higher percentage ofnon-hybrid seed.

In other aspects of the present invention, the method provides formaximization of seed yield at various hybridity levels.

In further aspects of the present invention, the method provides forseparate harvesting of intermingled crops.

In further aspects of the present invention, the method provides forverification of percentage of hybridity of a hybrid product.

These and further objects and advantages of the present invention willbecome clearer in light of the following detailed description of anillustrative embodiment of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The illustrative embodiment may best be described by reference to theaccompanying drawings where:

FIG. 1 shows a flow diagram of one embodiment of the methods forproducing a hybrid seed product.

FIG. 2 a shows a regression curve showing percentage hybridity versusfemale to pollenizer ratio for a single cross.

FIG. 2 b shows a regression curve showing percentage hybridity versusfemale to pollenizer ratio for multiple crosses.

FIG. 3 shows a plot planted according to the subrow method of theinvention.

All figures are drawn for ease of explanation of the basic teachings ofthe present invention only; the extensions of the figures with respectto number, position, relationship, and dimensions of the parts to formthe preferred embodiment will be explained or will be within the skillof the art after the following description has been read and understood.Further, the exact measurements and measurement proportions to conformto specific percentages, sizes, and similar requirements will likewisebe within the skill of the art after the following description has beenread and understood. Values provided are representative and are utilizedto facilitate the description of the preferred embodiment.

Where used in the various figures of the drawings, the same numeralsdesignate the same or similar parts. Furthermore, when the terms“upper,” “lower,” “side,” “end,” “bottom,” “first,” “second,”“laterally,” “longitudinally,” “row,” “column,” “array,” and similarterms are used herein, it should be understood that these terms havereference only to the structure shown in the drawings as it would appearto a person viewing the drawings and are utilized only to facilitatedescribing the illustrative embodiment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention provides methods for producing a hybrid seedproduct of bee-pollinated crops such as alfalfa and soybean. FIG. 1shows a flow diagram of one example embodiment of these methods. Themethod includes selection of a female line and a pollenizer line 10.These lines are planted in test plots in selected ratios of femaleplants to pollenizer plants 20. Data is collected from the test plots30. Data analysis provides information on characteristics such as: seedsize distribution for each line and seed yield of female and pollenizerplants in response to the selected ratios of female plants to pollenizerplants 40. In the preferred embodiments, the data analysis can providefor further selection of a female line or lines and further selection ofa pollenizer line or lines for production of the hybrid seed product. Inone example embodiment, the data analysis 40 may indicate a combinationof a female line and pollenizer line having a high seed yield.

In this example embodiment, analysis of seed size distributiondetermines a procedure for producing the hybrid seed product. If theseed size distribution of the female plants and the pollenizer plantshave a small amount of overlap 50, then a female to pollenizer ratio isselected using the data analysis to produce the maximum quantity of seed60. In one example embodiment, selection of a female to pollenizer ratioto produce the maximum quantity of seed occurs when the overlap in seedsize distribution involves less than 25% of the seed product of thecross between the selected female line and the selected pollenizer line.Subsequent seed size screening removes enough non-hybrid seed to achievea targeted percentage of hybridity in the hybrid seed product. Wherethere is little or no overlap in seed size distribution, substantiallyall of the non-hybrid seed may be removed 70.

If the seed size distribution of the female plants and seed sizedistribution of the pollenizer plants have significant overlap, but arenot substantially the same 80, then statistical analysis on therespective seed size distributions determines the percentage ofhybridity that can be gained by screening out seed within a selectedsize range 90. Selection of a female to pollenizer ratio for aproduction field takes into account both the hybridity level of theselected female to pollenizer ratios in the test plots, and the gain inhybridity level attained by screening out seed in a selected size range100. In the preferred embodiment, the ratio producing the maximumquantity of seed after post-harvest screening to achieve a targetedhybridity level is selected for use in the production field.

If the seed size distribution of the female plants and the seed sizedistribution of the pollenizer plants are substantially the same 110,then a female to pollenizer ratio is selected for the production fieldusing data from the test plots that will achieve the targeted hybriditylevel 120.

The selected female line and the selected pollenizer line are thenplanted in the production field in the selected female to pollenizerratio to produce the hybrid seed product 130. After production, thepercentage of hybridity of the hybrid seed product is verified 140.

With respect to the selection of lines 10 for the hybrid seed product,in the preferred embodiment of the methods of the invention, theproduction of a hybrid seed product includes selection of one or morefemale lines, also referred to as an “A line.” A female line is acytoplasmic male sterile line, a genic male sterile line, or an inducedmale sterile line by chemicals or biotechnology. These lines have acondition in which pollen is absent or non-functional in floweringplants. The methods also include the selection of one or more pollenizerlines, also referred to as a “male line” or “C line.” The pollenizerline is a male fertile line having a condition in which pollen isproduced and functional in flowering plants, with the female part eitherfunctional or not functional.

In the preferred embodiment, selection of the female line and thepollenizer line takes into account both agronomic factors and factorsthat affect percentage of hybridity and seed yield. In an exampleembodiment, these factors include female to pollenizer ratio, seed yieldfrom pollenizer plants, seed yield from female plants, female topollenizer seed yield differential, female to pollenizer seed sizedifferential, high seed yield with increasing female to pollenizer plantratio, low P.P.I. (Pollen Production Index), large seed size,pollination power in pollenizer plants and high self-compatibility inpollenizer plants. In further aspects of the invention, these factorsinclude desirable agronomic traits such as disease resistance, insectresistance and forage yield.

The selected lines can then be planted in test plots 20 for datacollection 30 and data analysis 40. In the preferred embodiment, data onseed yielding ability, pollination power, pollen production index(P.P.I.), seed size distribution for each line and seed yield of femaleand pollenizer plants in response to the selected ratios of femaleplants to pollenizer plants is collected along with othercharacteristics.

The data collection 30 and data analysis 40 of the present inventiondemonstrate that individual female lines can have different seedyielding abilities. Table 1 illustrates test results of seed yield forfour female lines with different P.P.I., with each female line crossedwith five different pollenizers.

TABLE 1 Seed Yield of Female Plants with Different P.P.I. Female LineAverage seed Yield (AXB) P.P.I. (Total wt. of 4 rows in grams) Female10.013 328.6 Female2 0.025 402.1 Female3 0.046 318.2 Female4 0.085 369.6

Table 1 illustrates the results of testing under the followingconditions:

-   -   Selection of four female lines with different P.P.I. and five        pollenizer lines with different pollinating abilities for        diallel mating in three female to pollenizer ratios, 70:30,        75:25 and 80:20 at two locations, Sloughouse, Calif., and        Homedale, Id. p1 Split plot designs with two replications were        used at the two locations. Each location included 120 plots,        with each plot having six rows and being 20 feet long. The        center four rows included a mixture of both female and        pollenizer seed in one of the tested ratios. The outside rows        included only female seed and provided for P.P.I., readings and        progeny testing.

These four female lines demonstrate different seed yieldingcapabilities. As further shown in Table 1, experimentation reveals nosignificant correlation between P.P.I. and seed yields for the testedfemale lines in the tested female to pollenizer ratios within the testedlow P.P.I. range. In this experiment, each female line was crossed withfive male lines in three female to pollenizer ratios. Thus, in apreferred embodiment of the invention, the methods of the invention forhybrid seed production includes selection of at least one female linehaving a high seed yield, such as the line designated Female2 in thestudy above, without regard for the P.P.I. of the female line. In otheraspects of the present invention, the invention may employ multiplefemale lines having varying seed yield. Those skilled in the art willrecognize that selection of a female line may be done according tovarious agronomic factors within the spirit and scope of the invention.

In the preferred embodiment of the present invention, selection of apollenizer, or male, line provides for high pollination power. Thus, ameasure of pollination power provides a measure of average female lineseed yield from pollination of the selected pollenizer line. Tables 2and 3 show representative measures of pollination power for five malelines.

TABLE 2 Seed Yield of Pollenizers Pollenizer Seed Yield (grams/plant)Male1 361.5 Male2 333.5 Male3 360.8 Male4 440.7 Male5 276.6

Table 2 shows data collected according to the teachings of the presentinvention on average seed yield for five pollenizers crossed with fourdifferent females in three female to pollenizer ratios: 80:20, 75:25 and70:30. Seed were harvested in bulk, comprising a mixture of both hybridand pollenizer seed. Seed yields axe the mean of twenty-four plots. TheMale5 pollenizer line had the white flower trait.

Testing according to the teachings of the present invention indicatesthat different pollenizers have different seed yielding abilities andvarying pollinating power with respect to female plant seed yield. Inthe study performed above, the Male4 pollenizer caused the female plantsto produce the most seed, and the Male5 pollenizer caused the femaleplants to produce the least seed. These results are statisticallysignificant at the 1% level.

TABLE 3 Average Seed Yield of Each Female with Specific Pollenizer(Male) at Three Female to Male Ratios (70:30, 75:25, and 80:20) MaleMale4 Male5 Female Male1 Male2 Male3 (High seed) (Low seed) Female1316.8 339.2 316.8 383.8 286.7 Female2 431.2 392.5 361.5 534.3 290.8(high seed yielder) Female3 297.0 298.5 358.0 402.3 235.3 (low seedyielder) Female4 401.2 303.7 406.8 442.8 293.5

Testing according to the teachings of the present invention confirm thatcrossing females having high seed yield with pollenizers having highseed yield results in a cross with the highest seed yield. Likewise,crossing females having low seed yield with pollenizers having low seedyield results in a cross with the lowest seed yield. Table 3 summarizesthe results of this testing.

According to the further teachings of the present invention, thepercentage of hybridity of the hybrid seed product will be highest whenusing a male line with high pollination power but low seed yield. Thisprovides for a higher percentage in the production field of hybrid seedby lowering production of non-hybrid seed. In the preferred form of theinvention, the method includes selection of a male line with low seedyield but high pollination power for crossing with a female line withhigh seed yield in hybrid seed production. In another aspect of thepreferred embodiment of the invention, pollenizers having highself-incompatibility contribute to a hybrid seed product having lessselfed seed than outcross seed. As with selection of the female line, inother aspects of the present invention, the method of the invention mayemploy multiple pollenizer lines having varying pollination power. Thoseskilled in the art will recognize that selection of a pollenizer linehaving lower pollination power and lower self-incompatibility is withinthe spirit and scope of the invention, and that selection of apollenizer line may be done according to various agronomic factors.

TABLE 4 Seed Yield Decrease with Increase Female and Decrease Pollenizer(Male) in Female to Male Ratios Female:Pollenizer(male) 70:30 75:2580:20 Seed Yield 375.2 370.1 318.6 (Hybrid seed and pollenizer seed) %reduction from 70:30 1.4 15.1 seed production level

In the most preferred embodiment of the invention, the method forproduction of hybrid seed further incorporates selection of female topollenizer ratios. Testing according to the teachings of the presentinvention demonstrates that female to pollenizer ratio affects seedyield, as shown in Table 4. This testing further indicates that theeffect of female to pollenizer ratio is not linear. In one exampleembodiment, experimentation on selected female and pollenizer linesdemonstrates an average 1.4% drop in seed yield between a 70:30 and a75:25 female to pollenizer ratio. However, testing demonstrated anaverage 15.1% drop in seed yield between a 75:25 and an 80:20 female topollenizer ratio. Therefore, for the tested lines, much greater gains inseed yield occur when going from an 80:20 female to pollenizer ratio toa 75:25 ratio than from a 75:25 ratio to a 70:30 ratio. Table 4summarizes the average decreases in seed yield with increasing female topollenizer ratios for the tested varieties. Furthermore, testingaccording to the teachings of the present invention further demonstratesthat the decrease in seed yield will vary with different female topollenizer crosses, as shown in Tables 6,7,8, and 9.

According to the teachings of the present invention, individual crosseshave varying responses to female to pollenizer ratio with respect tohybrid seed production levels and percentages of hybridity. Thus, in thepreferred form of the invention, production of a hybrid seed productincludes determining, for each cross, the hybrid seed production leveland percentage of hybridity for a range of female to pollenizer ratios.In this preferred embodiment, hybrid seed production employs selectionof a specific female to pollenizer ratio for maximum seed production ata desired hybridity level for each cross of two or more lines. However,those skilled in the art will recognize that selection of a female topollenizer ratio can be made for a selected cross without determiningthe hybrid seed production level and percentage of hybridity for a rangeof female to pollenizer ratios. For example, in one alternateembodiment, a typical response to female to pollenizer ratio may be usedfor production purposes without determining individual response tofemale to pollenizer ratio. In another alternate embodiment, wherenon-hybrid seed are removed to produce the hybrid seed product, a femaleto pollenizer ratio having high hybrid seed production levels, such as70:30 or 60:40 is selected for production.

TABLE 5 Seed Yield of a Female Line Pollinated with High and Low SeedYield Pollenizers at Three Different Female to Pollenizer Ratio'sFemale:Male ratio 1:1 2:1 3:1 total C1/C2 ratio A1 × C1/A1 × C2 ratioF(A) (wt/plant in grams) 59.9 48.6 38.9 147.4 M C1 (High yield) 45.237.4 39.5 122.1 122.1/35.3 147.4/46.1 (wt/plant in grams) % Hybridity(56.9) (72.2) (74.8) F(A)(wt/pl) in grams 17.4 15.3 13.4 46.1 M C2 (Lowyield) 12.9 11.8 10.6 35.3 (wt/plant in grams) Avg. Female seed wt. 38.732.0 26.2 Ave. Male seed wt. 29.1 24.6 25.1 % Hybridity (62.5) (73.1)(79.9)

In one preferred embodiment of the method of the invention to produce ahybrid seed product, selection of a cross having a high female topollenizer seed yield differential provides for an increase in thepercentage of hybridity of the hybrid seed product. The teachings of thepresent invention demonstrates that seed yield of female plants variesin response to different males at different female to pollenizer ratios.Table 5 illustrates results of seed yield for a female line crossed witha high seed yield pollenizer and a low seed yield pollenizer at threedifferent female to pollenizer ratios. Thus, greater levels of hybriditycan be obtained by selection for a cross where the pollenizer lines andfemale lines have a high female to pollenizer seed yield differential.In the preferred embodiment, the methods for producing a hybrid seedproduct of the instant invention include selection of a female line anda pollenizer line to provide for a high female to pollenizer seed yielddifferential.

The present invention further illustrates, as shown in Table 5, thatfemale seed yield has a positive correlation with decreasing female topollenizer ratios. Total seed yield also has a positive correlation withdecreasing female to pollenizer ratios. In contrast, the percentage ofhybridity has a positive correlation with increasing female topollenizer ratios. Female seed yield is also affected by pollenizer seedyield, with higher pollenizer seed yield positively correlated to higherfemale seed yield.

TABLE 6 Female1 with Male1 Seed Yield Decrease and Hybridity Increase asFemale to Male (Pollenizer) Ratios Changed from 1:1 Ratio to 2:1 Ratioand 3:1 Ratio Female:Male 1:1 2:1 3:1 F1 (wt/pl) grams 59.9 48.6 38.9 %reduction from 1:1 18.9 35.1 seed production level M1 (wt/pl) grams 45.237.4 39.5 % reduction from 1:1 17.3 12.6 seed production level Total(Female + Male) 105.1 86.0 78.4 % reduction from 1:1 18.2 25.4 seedproduction level F:M seed yield differential 1.33 1.30 0.98 % Hybridity57.0 72.2 74.7

TABLE 7 Female1 with Male2 Seed Yield Decrease and Hybridity Increase asFemale to Male (Pollenizer) Ratios Changed from 1:1 Ratio to 2:1 Ratioand 3:1 Ratio Female:Male 1:1 2:1 3:1 F1 (wt/pl) grams 17.4 15.3 13.4 %reduction from 1:1 12.1 23.0 seed production level M2 (wt/pl) grams 12.911.8 10.6 % reduction from 1:1 8.5 17.8 seed production level Total(Female + Male) 30.3 27.1 24.0 % reduction from 1:1 10.6 20.8 seedproduction level F:M seed yield differential 1.35 1.30 1.26 % Hybridity62.5 73.1 79.9

TABLE 8 Overall changes in Seed Yield Decrease and Hybridity Increase asFemale to Male (Pollenizer) Ratios Increases from 1:1 Ratio to 2:1 Ratioand 3:1 Ratio Female:Male 1:1 2:1 3:1 F1 (wt/pl) grams 59.9 48.6 38.9Change 100.0 81.1 64.9 M1 (wt/pl) grams 45.2 37.4 39.5 Change 100.0 82.787.4 Total (Female + Male) 105.1 86.0 78.4 Change 100.0 81.8 74.6 F:Mseed yield differential 1.33 1.30 .99 % Hybridity 56.9 72.2 74.8

Tables 6, 7 and 8 show data for a female line crossed with twopollenizer lines using three different female to pollenizer ratios.Table 6 shows the female line crossed with a first pollenizer line(Male1) at three female to pollenizer ratios. Table 7 shows the samefemale line crossed with, a second pollenizer line (Male2) at threefemale to pollenizer ratios. Data obtained from experimentationaccording to the teaching of the present invention demonstrates anaverage hybridity level of 71.8% for the cross with the first pollenizerline and an average hybridity level of 67.8%. for the cross with thesecond pollenizer line. Thus, according to the teachings of the presentinvention, percentage of hybridity will vary from cross to cross.

This experimentation further shows differences in response to seed yieldand percentage of hybridity from cross to cross. Table 6 shows that forthe first pollenizer (Male1), seed yield increased by 34% and 10% whenfemale to pollenizer ratio changed from 3:1 to 1:1 and 3:1 to 2:1,respectively. Percentage of hybridity, conversely, increased by 31% and27% when female to pollenizer ratios changed from 1:1 to 3:1 and 1:1 to2:1, respectively. Table 7 shows that for the second pollenizer (Male2),seed yield increased by 26% and 13% when female to pollenizer ratiochanged from 3:1 to 1:1 and 3:1 to 2:1, respectively. The percentage ofhybridity increased by 28% and 17%, conversely, when female topollenizer ratios changed from 1:1 to 3:1 and 1:1 to 2:1, respectively.Thus, according to the teachings of the present invention, in thepreferred embodiment of the invention for producing a hybrid seedproduct, each cross is tested for responsiveness to female to pollenizerratio for seed yield and percentage of hybridity. In the preferredembodiment, a cross is selected that maintains at least two-thirds offemale seed yield when increasing ratio of female plants to pollenizerplants from 1:1 to 3:1 under substantially similar environmentalconditions. Alternatively, for some varieties, the method for producinga hybrid seed product can make use of a generic formula to estimateresponsiveness to female to pollenizer ratio tor seed yield andpercentage of hybridity. One way of establishing a generic curve is byestimating a curve from multiple crosses of other varieties. FIG. 2Ashows one example embodiment of a curve estimated from severalfemale-pollenizer crosses.

TABLE 9 The effect on hybrid seed yield and percentage of hybridity bychanging female to pollenizer ratios. Female to Pollenizer Ratios.Female 70:30 75:25 80:20 F1 SEEDYIELD 345 298 218 % CHANGE 100 86 63HYBRIDITY 87.5 93.5 90.5 % CHANGE 100 107 103 F2 SEEDYIELD 285 357 231 %CHANGE 100 125 81 HYBRIDITY 88.5 84.5 87.5 % CHANGE 100 96 99 F3SEEDYIELD 340 174 193 % CHANGE 100 51 57 HYBRIDITY 84.5 88 91 % CHANGE100 104 108 F4 SEEDYIELD 344 285 252 % CHANGE 100 83 73 HYBRIDITY 83.588.00 89.00 % CHANGE 100 105 107 OVER ALL SEEDYIELD 100 86 69 CHANGEHYBRIDITY 100 103 104

Table 9 summarizes further experimental seed yield and percentage ofhybridity tor multiple female and pollenizer crosses at different femaleto pollenizer ratios. In the most preferred embodiment, production of ahybrid seed product includes a statistical analysis on data for aselected cross in order to determine the effect of different female topollenizer ratios on seed yield. In one example embodiment, thestatistical analysis comprises a regression analysis. FIG. 2B shows anumber of regression curves for different female-pollenizer crosses.

As shown in Table 9, according to the teachings of the presentinvention, changing female to pollenizer ratios from a 70:30 ratio to a75:25 ratio or a 70:30 ratio to an 80:20 ratio greatly impacts hybridseed yield. Seed yield dropped an average of 31% from a 70:30 ratio to80:20 ratio and dropped an average of 16% from a 70:30 ratio to a 75:25ratio, respectively, when Male3 was crossed with four different femalelines. Average percentage of hybridity increased 3% when female topollenizer ratios changed from a 70:30 ratio to a 75:25 ratio andincreased 4% when going from a 70:30 ratio to an 80:20 ratio,respectively.

While a hybrid seed product having a high percentage of hybridity isdesirable for agronomic qualities and for certification; on the otherhand, the cost of producing a hybrid seed product increases withdecreasing seed yield. The methods for producing a hybrid seed productof the present invention, therefore, provides methods for determining afemale to pollenizer ratio providing for maximum seed yield at apredetermined level of hybridity of the hybrid seed product.

TABLE 10 Hybrid Seed Yield and Percentage of Hybridity are Differentwith Each Specific Combination of Female Line and Pollenizer atDifferent Female to Pollenizer Ratios Female seed % Female seed F:M % ofMale. Hybridity yield Change % F1:M1 0.7:1.0  78.7/100.0 44.0 3:161.6/100 64.9 Female ratio change. (0.7:1.00 to 3:1) (78.3%) F2:M21.1:1.0  92.3/100.0 48.0 3.2:1.0 65.1/100 75.3 Female ratio change.(1.1:1.0 to 3.2:1.0) (70.5) F3:M3 1:1 141.0 58.5 2.5:1  97.8/100 71.0Female ratio change. (1:1 to 2.5:1) (69.4) F1:M4 0.8:1.0 101.4/100  50.33.0:1.0 115.1/100  77.5 Female ratio change (0.8:1.0 to 3:1) (113.5) 52.3:1.0 86.8/100 66.6 3.0:1.0  100/100 75.0 Female ratio change (2.3:1.0to 3:1) (115.2)

Table 10 summarizes experimentation according to the teachings of thepresent invention to determine varying effects of female to pollenizerratios on different crosses. In this example embodiment, Female1 (F1),Female2 (F2) and Female3 (F3) seed yield decreased at higher female topollenizer ratios when pollinated by Pollenizer1 (M1) and Pollenizer2(M2). Female4 (F4) and Female5 (F5), on the other hand, did not showseed yield decrease at higher female to male ratios when pollinated byPollenizer4 (M4) and Pollenizer5 (M5). With respect to the lines testedand reported on Table 9, (A1XB1) X (A2XR (Restorer)) cross demonstratesboth higher hybrid seed yield and higher percentage of hybridity.

In the preferred embodiment of the invention, the data analysis 40comprises a regression analysis of percentage of hybridity versus femaleto pollenizer ratio to establish a regression curve for each individualcross. In this embodiment of the present invention, selection of linesfor hybrid seed production employs the resulting regression curves foreach cross under consideration in order to select a cross for high seedyield during hybrid seed production. FIG. 2B shows a regression curveshowing percentage hybridity versus female to pollenizer ratio formultiple crosses. In this example embodiment, the regression analysisshows:

y=18.836ln(x)+55.128

where: y=percentage of hybridity

-   -   x=female to pollenizer ratio.

In this embodiment of the invention to produce a hybrid seed product,the regression analysis allows for determination of a female topollenizer ratio necessary to achieve a targeted percentage ofhybridity. In one example embodiment, selection of the female topollenizer ratio employs the regression analysis to target a 75%hybridity level to achieve certification standards for a hybrid variety.In an alternative embodiment, selection of the female to pollenizerratio employs the regression analysis to target a 95% hybridity level.

However, in many female to pollenizer crosses, seed yield dramaticallydecreases with increasing female to pollenizer ratios. Agronomic orother considerations often suggest a cross having a dramatic decrease inseed yield with increasing female to pollenizer ratios. Therefore, thepresent invention provides a means for post-production increase ofpercentage of hybridity in a hybrid seed product.

In the preferred embodiment of the invention, the method for productionof a hybrid seed product increases the percentage of hybridity of ahybrid seed product by removing non-hybrid seed. In one exampleembodiment, removal of non-hybrid seed makes use of seed sizedifferential between hybrid seed produced by female plants andnon-hybrid seed produced by pollenizer plants. In this preferredembodiment, selection of a female line and selection of a pollenizerline results in hybrid seed having an average size larger thannon-hybrid seed. In an alternate embodiment, selection of a female lineand selection of a pollenizer line results in hybrid seed having anaverage size smaller than non-hybrid seed.

Testing according to the preferred teachings of the inventiondemonstrates selection of a female line and a pollenizer line canprovide a cross having a high correlation (r=0.9) between femaleparental lines and their first generation progeny with respect to seedsize. Thus, in the preferred embodiment, the methods for producing ahybrid seed product employs selection for hybrid seed having a seed sizedistribution skewed to larger seed sizes and non-hybrid seed having aseed size distribution skewed to smaller seed sizes. Sifting productionseed to screen out seed smaller than a selected seed size increases thepercentage of hybrid seed for the hybrid seed product. In one exampleembodiment, a sieve screens out seed smaller than a selected seed sizeto increase the percentage of hybridity of the hybrid seed product. Inone example embodiment, shown in Table 14, using a 1/19″ sieve providesa gain to 75% hybridity when production of hybrid seed is done using a65:35 ratio of female plants to pollenizer plants. As those skilled inthe art will recognize, different sieve sizes can be employed toaccomplish different increases in percentage of hybridity.

TABLE 12 Seed Size Distributions of a Female Line, 3 Pollenizers and aHybrid (A1 × B1) × (C1 + C2 + C3) 1/14 1/15 1/16 1/17 1/18 1/19 1/201/21 1/22 1/23 REMAIN MEAN H1 (hybrid) 0.49 14.99 25.22 25.72 20.97 8.470.35 3.54 0.22 0.01 0.00 5.93 F1 (Female 1) 1.17 18.60 23.73 26.24 18.117.54 0.22 3.72 0.28 0.35 0.00 5.96 C1 (Pollenizer 1) 0.73 1.46 4.4715.56 29.18 21.97 2.09 21.49 2.58 0.19 0.29 5.37 C2 (Pollenizer 2) 0.562.08 7.21 21.01 21.86 24.32 1.91 19.14 1.80 0.12 0.00 5.45 C3(Pollenizer 3) 0.80 10.64 21.78 27.68 20.08 10.16 0.58 7.17 0.83 0.260.01 5.82 Mean of C1 + C2 + C3 0.70 4.73 11.16 21.41 23.71 18.82 1.5315.93 1.74 0.19 0.10 1,000 SEED WEIGHT H1 = (A1 × B1) × (C1, C2, C3)2.480 GRAMS F1 = A1 × B1 2.365 GRAMS C1 2.200 GRAMS C2 2.339 GRAMS C32.250 GRAMS

TABLE 13 Seed Size Distribution of a Female Line, 3 Pollenizers a HybridUsing a 1/19″ sieve to 1/23″ sieve Entry 1/19 1/20 1/21 1/22 1/23 Hybrid(A1XB1) 12.59% 4.12% 3.77% 0.23% 0.01% XC1, C2, C3) Female (AXB) 11.92%4.58% 4.36% 0.63% 0.35% Pollenizer (C1) 47.37% 26.93% 24.87% 2.87% 0.23%Pollenizer (C2) 46.26% 23.38% 21.47% 1.96% 0.12% Pollenizer (C3) 18.54%8.90% 8.32% 1.11% 0.27% Pollenizer Average 37.39% 19.74% 18.22% 1.98%0.21%

As shown in Table 12, seed size differential varies for each crossing ofdifferent female lines and pollenizer lines. Table 12 shows a seed sizedistribution for a hybrid line, a female line, and three pollenizerlines. Table 13 shows the percentage of seed screened out using fivedifferent sieve sizes, from 1/19 to 1/23.

According to the teachings of the present invention, seed sizedistributions vary for each different cross; therefore, increases inhybridity level also vary with each different cross between differentfemale lines and different pollenizer lines. Because seed size is ainheritable trait, in the preferred embodiment of the present inventionfor producing a hybrid seed product, selection of female and pollenizerlines provides for seed size differential between hybrid and non-hybridseed. Environmental conditions, such as drought and poor soilconditions, also affect seed size. Thus, determination of seed sizedistribution according to the teachings of the present invention, shouldbe done in the same field to control for environmental variation. As theseed size distribution differential between female line and pollenizerline increases, the environmental effect on the use of screening toremove non-hybrid seed will be reduced. Experimentation according to theteaching of the present invention, and as shown in Table 14, illustratesthe increase in hybridity levels achieved by straining out a selectedseed size for crosses between one female line and three pollenizerlines. In particular, Tables 14a and 14b illustrate gains in hybriditylevel for a 1/19″ sieve and a 1/21″ size sieve for the selected crosses.

Percentage of Hybridity Gained by Applying 1/19″ Sieve and 1/21″ Sieveto Screen Out a Higher Percentage of Non-Hybrid Seed (Pollenizer C₁, C₂,C₃)

TABLE 14a AMT. SEED SCREEN FEMALE: % *% HYBRID PARENTS OUT FOR 1/19 SIZEPOLLENIZER HYBRIDITY FOR FEMALE POLLENIZER FEMALE POLLENIZER RATIO INC.PRODUCT A1 × B1 C1 11.92% 47.37% 65:35 10.7 75.7 C2 46.26% 10.3 75.3 C318.54% 1.8 66.8 C1 70:30 9.6 79.6 C2 9.3 79.3 C3 1.6 71.6 C1 75:25 8.483.4 C2 8.1 83.1 C3 1.4 76.4 C1 80:20 7.0 87.0 C2 6.8 86.8 C3 1.2 81.2

TABLE 14b AMT. SEED SCREEN FEMALE: % % HYBRID PARENTS OUT FOR 1/21 SIZEPOLLENIZER HYBRIDITY FOR FEMALE POLLENIZER FEMALE POLLENIZER RATIO INC.PRODUCT A1 × B1 C1 4.36% 24.87% 65:35 5.3 70.3 C2 21.47% 4.3 69.3 C38.32% 1.0 66.0 C1 70:30 4.8 74.8 C2 4.0 74.0 C3 0.9 70.9 C1 75:25 4.379.3 C2 3.5 78.5 C3 0.8 75.8 C1 80:20 3.6 83.6 C2 3.0 83.0 C3 0.7 80.7

In alternate embodiments of the invention, the method forpost-production increase of percentage of hybridity can employdifferences in seed density, seed weight or seed coat color. In oneexample embodiment, selection of a female line and a pollenizer line mayprovide for non-hybrid seed having a lesser density than hybrid seed. Inthis example embodiment, the teachings of the present invention providesfor removal of non-hybrid seed according to seed density or seed weight.A gravity table or rice roller can separate hybrid and non-hybrid seedin this embodiment of the invention. In an alternate embodimentemploying differences in seed color between hybrid seed and non-hybridseed, a seed coat color sorter removes non-hybrid seed. Those skilled inthe art will recognize that other methods of removing non-hybrid seed toincrease the percentage of hybridity of a hybrid seed product lie withinthe spirit and scope of the invention. For example, morphological traitssuch as seed size, seed color, seed weight, leaf shape, leaf size, rootcolor, stem color, flower color root structure, plant height and fallgrowth habit or a genetic marker can be used to distinguish hybrid seedfrom non-hybrid seed.

Therefore, in the preferred embodiment of the invention to producecertified seed, the regression curve of a targeted hybrid productprovides for selection of a female to pollenizer ratio, that whencombined with a post-production increase in hybridity level, achievesmaximum yield at a hybridity level of at least 75%.

In the preferred embodiment of the invention to produce a hybrid seedproduct, test plots provide for determination of percentage of hybridityat different female to pollenizer ratios. Regression analysis ofpercentage of hybridity versus female to pollenizer ratio then providesa regression curve for each individual cross to allow for production ofa hybrid product having a preselected percentage of hybridity. In otheraspects of the method of the invention, the test plots further providefor determination of the seed size distribution of selected femalelines, male lines and hybrid lines. The test plots also provide fordetermination of the 1,000 seed weight of each line and the P.P.I. ofthe female lines.

In one example embodiment of the invention to produce a hybrid seedproduct, the following types of test plots are employed.

A “Check” plot has female seed and pollenizer seed planted in the sameplot. The check plot includes two pollenizer rows, a skipped row, fourfemale rows, a skipped row, and two pollenizer rows. This design of the“Check” plot allows the female rows and pollenizer rows to be harvestedseparately. After flowering, analysis of the female plants provides fordetermination of the P.P.I. After harvesting, analysts of the seed fromthe female plants provides determination of seed size distribution and1000 seed weight. After harvesting the pollenizer plants, analysis ofthe seed from pollenizer plants provides determination of seed sizedistribution and 1000 seed weight. Analysis also determines data for theseed yield of female plants and pollenizer plants.

A female to pollenizer plot designed to have a 1:1 ratio. The female andpollenizer plants are intermingled and distributed randomly.

A female to pollenizer plot designed to have a 2:1 ratio. The female andpollenizer plants are intermingled and distributed randomly.

A female to pollenizer plot designed to have a 3:1 ratio. The female andpollenizer plants are intermingled and distributed randomly.

A female to pollenizer plot designed to have a 4:1 ratio. The female andpollenizer plants are intermingled and distributed randomly.

In one preferred embodiment, the test plots are planted using a completerandom block design with four to eight replications for each female topollenizer ratio. In this embodiment, the test plots are six row plots,with each row fifty to one hundred feet long. Data collection isperformed on the middle two to four rows, with the outside rows servingas borders. The outside border rows serve as a guard and a barrier toreduce contamination from neighboring plots.

In this example embodiment of the invention, data collection follows thefollowing procedures. The number of seed produced by female plants canbe determined using the equation:

x=female seed weight/weight of one thousand female seed,

where x=the mean number of seed produced by a female plant.

The number of seed produced by pollenizer plants can be determined usingthe equation:

y=pollenizer seed weight/weight of one thousand pollenizer seed,

where y=the mean number of seed produced by a pollenizer plant.

The percentage of female seed can be then determined as:

% female seed=x/(x+y).

In the preferred embodiment of the invention, determination ofpercentage of hybridity takes selfing and sibbing in the female lineinto account. The adjusted percentage of hybridity corrects for thesefactors by subtracting non-hybrid seed produced from female plants. TheASOCA publication of hybrid alfalfa certification provides thecorrection factor for non-hybrid seed produced from female plants fromsibbing and selfing. The correction factor is equal to P.P.I.×0.595,with the P.P.I. (Pollen production index) defined as follows.

1. Male Sterile Plants (MS) P.P.I.=0

-   -   No visible pollen can be observed with the naked eye when flower        is tripped with a black knife blade.

2. Partial Male Sterile Plants (PMS) P.P.I.=0.1

-   -   A trace of pollen can be observed with the naked eye when flower        is tripped with a black knife blade.

3. Partial Fertile Plant (PF) P.P.I.=0.6

-   -   Less than normal amount of pollen can be observed with the naked        eye when flower is tripped with a black knife blade.

4. Fertile Plant (P) P.P.I.=1.0

-   -   Normal amounts of pollen can be observed when flower is tripped        with a black knife blade.

In this example embodiment of the invention, the pollen production index(P.P.I.) is determined by sampling 200 female plants from the femalerows of the “check” plots. The percentage of hybridity can then bedetermined by the equation:

% hybridity=% female seed−% non-hybrid female seed,

where the % non-hybrid female seed is P.P.I.×0.595

In one preferred embodiment of the invention, the methods for producinga hybrid seed product determines the percentage of hybridity for eachfemale to male ratio of 1:1, 2:1, 3:1, and 4:1,

This method for determining the percentage of hybridity requiresanalysis of seed produced by female plants apart from analysis of seedproduced by pollenizer plants. Harvesting female plants separately frompollenizer plants is one way of obtaining female seed apart frompollenizer seed. However, separate harvesting of female plants frompollenizer plants is difficult under current hybrid seed productionmethods.

In one method for hybrid seed production, female plants are crossed withpollenizer plants by planting female plants and pollenizer plants inseparate rows of plants, with each row separated by a width of twenty toforty inches. Each group of rows contain either pollenizer plants orfemale plants, such as shown in U.S. Pat. No. 4,045,912, hereinincorporated by reference. This allows female plants and pollenizerplants to be harvested separately according to rows. However, thismethod involves a large physical separation of female and pollenizerplants, resulting in lower production of hybrid seed.

Female and pollenizer plants may also be intermingled in the same row inthe same test plot. This intermingling increases hybrid seed productionfrom the female plants. However, intermingling also causes difficulty indistinguishing the female plants from the pollenizer plants. To obtaindata on hybrid seed production, seed from female plants must bedistinguished from seed from pollenizer plants. Distinguishing betweenfemale plants and pollenizer plants is a highly labor intensive,difficult and expensive process. U.S. Pat. No. 4,045,912 provides oneexample of such a process, where female and pollenizer plants aregerminated separately, and then hand planted and tagged in a test plot.This process becomes untenable in larger applications. Thus, the presentinvention provides methods for distinguishing female plants frompollenizer plants suitable for large-scale applications, such as largetest plots.

The preferred embodiment of the invention for producing a hybrid seedproduct provides for methods for distinguishing female plants frompollenizer plants by planting female lines and pollenizer lines insubrows. The subrow methods of the invention further allow for separateharvesting of female plants and pollenizer plants. During planting,female plants and pollenizer plants are offset from the center of a rowon opposite sides to distinguish the female plants from the pollenizerplants.

FIG. 3 shows an example embodiment of the subrow planting method of theinvention. The subrow planting method provides for planting both femaleand pollenizer plants in a single row 200, with female seed planted twoto four inches from the center 210 of the row 200 on a first subrow 220,and pollenizer seed are planted one to eight inches from the center 210of the row 200 in a second subrow 230 on the opposite side. Thus, inthis example embodiment, planting according to the subrow methodsprovides female plants 240 offset to one side in the first subrow 220and a pollenizer plants 250 in the second subrow 220 offset to the otherside. This single row 200 shows an embodiment having a female topollenizer ratio of 2:1. A second single row 260 shows an embodimenthaving a female to pollenizer ratio of 3:1. A third single row 270 showsa female to pollenizer ratio of 4:1. In one preferred embodiment of theinvention, an onion planter may be used to plant female seed andpollenizer seed in their respective subrows. Those skilled in the artwill recognize that other methods of planting the female seed and thepollenizer seed in their respective subrows lies within the spirit andscope of the invention.

In other aspects of the subrow methods of the invention, the subrows220, 230 can advantageously provide for reduction of competition betweenfemale plants and male plants by increasing physical separation of thefemale plants from the pollenizer plants while still allowingintermingling. In cases where one line may be more vigorous thananother, the increased separation between plants may allow slowergrowing plants a better opportunity to grow without being crowded out byother plants. Because of the small seed size of some field crops, suchas alfalfa, during planting, some seeds are not spaced with sufficientroom to grow, allowing a faster growing plant to crowd out a slowerdeveloping plant, particularly in subsequent years of perennial crops.Subrows provide for increased separation between female plants andpollenizer plants that reduces competition between the female plants andpollenizer plants, maintaining a more consistent female to pollenizerratio. As the female to pollenizer ratio is selected to optimizeproduction of the hybrid seed product, in this aspect of the presentinvention, planting in subrows in the production field maintains optimalproduction from year to year.

In another aspect of the subrow methods of the invention, subrows 220,230 allow a breeder to quickly determine the actual female to pollenizerratio. At a seedling stage, the subrows 220, 230 allow the number offemale plants 240 and the number of pollenizer plants 250 to be quicklycounted. In subsequent years after planting for perennial plants, a newfemale to pollenizer ratio needs to be determined from year to year, asplants may die over the winter. In this instance, subrows 220, 230facilitate quick determination of female to pollenizer ratio throughvisual inspection.

In another aspect of the invention, subrows 220, 230 facilitate separateharvesting of female plants 240 and pollenizer 250 by harvestingaccording to female subrows and pollenizer subrows. Separate harvestingallows for determination of percentage of hybridity by sampling beforeharvest.

In an alternate embodiment of the invention, morphological markers, suchas seed size, seed color, seed weight, leaf shape, leaf size, rootcolor, stem color, flower color root structure, plant height and fallgrowth habit flower colors, can be used to distinguish female plantsfrom pollenizer plants in the test plots. Those skilled in the art willrecognize that other methods of distinguishing female plants frompollenizer plants can be employed without departing from the spirit andscope of the invention.

The preferred form of the invention further includes verification ofpercentage of hybridity of the hybrid seed product 140. These methodsinvolve progeny testing and are employed after production of the hybridseed product in the production field. Verification of percentage ofhybridity can be used to help ensure a hybrid seed product achievespredetermined hybridity goals. In the preferred embodiment, acharacteristic of the hybrid seed that has a distinct difference fromnon-hybrid seed is used in the verification process. However, as thoseskilled in the art will understand, other methods for verifyingpercentage of hybridity can be employed without departing from thespirit or scope of the invention.

In one preferred form, verifying percentage of hybridity begins withchoosing a row and sampling 200-1000 female plants and 200-1000pollenizer plants. Data is collected to determine the seed yield of thefemale plants and the seed yield of the pollenizer plants. The female topollenizer ratio is determined by counting number of female plants tonumber of pollenizer plants for 200 feet in a chosen row for two to fivereplications in a hybrid seed production field. The subrow system can beemployed to aid tracking of female plants versus the pollenizer plantsto distinguish the plants in the commercial hybrid seed productionfield.

Analysis on the gathered data then provides an estimated percentage ofhybridity. Because of the variability inherent in biological systems,the analysis provides an estimate rather than an exact determination.The analysis can be performed according to the following procedures:

1. From the sampled plants, determine the average female seed yield (X)to pollenizer seed yield (Y) per plant and determine average number ofseed produced from female (hybrid) plants to number of seed producedfrom pollenizer plants as X:Y.

2. Determine number of female plants to number of pollenizer plants in acommercial hybrid seed production field by measuring the number offemale plants and number of pollenizer plants in 100 feet of a randomlyselected row with two to five replications. The proportion of femaleplants to the number of pollenizer plants is A:B.

3. The percentage of hybridity is computed according to:

${\% \mspace{14mu} {Hybridity}} = {{\frac{\left( {A \times X} \right)}{\left( {A \times X} \right) + \left( {B \times Y} \right)} \times 100\%} - {CF}}$

where CF is a correction factor to account for non-hybrid seed producedfrom female plants by selfing, and is equal to the P.P.I. (femaleplants)×0.595.

Harvesting the center two rows of pollenizer line from the pollenizertest strip and harvesting the center two rows of the female line fromthe female test strip separately provides for data on seed sizedistribution of the pollenizer seed and hybrid seed. The commercialhybrid production field is harvested in bulk.

In another example embodiment, seed size distribution differentialbetween hybrid seed and pollenizer seed provides data for verifyingpercentage of hybridity. In this method, four to six rows of 200-1000feet of plants from a selected pollenizer line are planted as apollenizer test strip and four to six rows of 200-1000 feet of plantsfrom a selected female line are planted as a female test strip in thesame commercial hybrid seed production field.

Analysis of the pollenizer seed provides the pollenizer seed sizedistribution. Likewise, analysis of the female seed, provides the femaleseed size distribution, and analysis of the bulk-harvested seed providesthe seed size distribution of the commercial hybrid production field. Byway of illustration and not limitation, the following shows an examplewith the seed size distribution determined for each increment from 1/10inch to 1/25 inch. In this example, samples having the same weight areused to determine the seed size distributions. The following procedureprovides an estimate of the percentage of hybridity.

1. Perform a T-test for seed size distribution of pollenizer seed,hybrid seed and commercial experimental hybrid seed (female:pollenizerratio block used by commercial hybrid seed production field) harvestedfrom a test plot, and comparing with pollenizer seed, hybrid seed andcommercial hybrid seed harvested from a commercial hybrid seedproduction field.

2. If the T-test is not significant, then the percentage of hybrid seedin the overlap area of the seed size distribution in the test plot wouldbe same as overlap area of the seed size distribution in the commercialhybrid production seed field.

The % hybridity in the commercial hybrid production seed field can thenbe determined by:

${{\% \mspace{14mu} {Hybridity}} = {{\frac{{X\; 1} + {Y\; 2}}{{X\; 1} + {Y\; 2} + {Q\; 2} + {R\; 1}} \times 100\%} - {CF}}}\;$

Where:

X=Hybrid seed weight of non-overlap area.

X1=X/1,000 seed weight of non-overlap area.

Y=Hybrid seed weight and pollenizer seed weight of overlap area.

Y1=Y×% Hybrid seed overlap area (from test plots).

Y2=Y1/1,000 seed weight.

Q=Pollenizer seed weight of overlap area.

Q1=Y×% Pollenizer seed of overlap area.

Q2=Q/1,000 seed weight.

R=Pollenizer seed weight of non-overlap area.

R1=R/1,000 seed weight.

CF=P.P.I. (female plants)×0.595.

In an alternate embodiment, seed size distribution can be used to verifypercentage of hybridity using the proportion of seed within a selectedseed size range. The selected seed size range includes an area ofoverlap in the seed size distribution between the seed size distributionof the female seed and pollenizer seed. The percentage of hybridity canbe estimated as:

${{\% \mspace{14mu} {Hybridity}} = {{\frac{Z - Y}{X - Y} \times 100\%} - {CF}}}\;$

where:

X=percentage of female seed fatting into the selected seed size range;

Y=percentage of pollenizer seed falling into the selected seed sizerange;

Z=percentage of production seed falling into the selected seed sizerange; and

CF=correction factor to account for non-hybrid seed produced from femaleplants by selfing, and is equal to the P.P.I. (female plants)×0.595.

As those skilled in the art will recognize, other seed characteristicswhere the female seed has a distinct difference from the pollenizer seedcan be used to estimate the percentage of hybridity. For example, in analternate embodiment, the mean seed size of the seed sampled from thepollenizer plants, the female plants and the production field can beused to estimate percentage of hybridity. Using this method, percentageof hybridity can be estimated as:

${{\% \mspace{14mu} {Hybridity}} = {{\frac{Z - Y}{X - Y} \times 100\%} - {CF}}}\;$

where:

X=mean female seed size;

Y=mean pollenizer seed size;

Z=mean production seed size; and

CP=correction factor to account for non-hybrid seed produced from femaleplants by selling and sibbing, equal to the P.P.I. (femaleplants)×0.595,

In another example embodiment, the methods for verification, ofpercentage of hybridity employ the pollen production index (P.P.I.) ofhybrid seed and the commercial hybrid seed product, which is a mixtureof hybrid and pollenizer seed. In this embodiment of the inventionemploying the P.P.I., verification of percentage of hybridity beginswith crossing a selected female line with a selected pollenizer line andharvesting the seed produced by the cross in a test plot and productionfield. The harvested seed is planted and P.P.I. data is gathered.Further analysis for verification of hybridity proceeds in one of twocases, depending on whether the P.P.I. of the hybrid seed productionfield is larger than the P.P.I. of the test plot hybrid seed or viceversa.

CASE 1

If the P.P.I. of the hybrid seed production field is larger than theP.P.I. of the test plot hybrid seed, the following formula will be usedto calculate the percentage of hybridity.

The seed from a hybrid seed production field contain both hybrid seed(female X pollenizer) and non-hybrid seed (pollenizer). The seed fromtest plot of f:p=1:0 contains only hybrid seed.

a. % of hybridity=1-P.P.I. (Production seed)/1-P.P.I. (Test plot hybridseed)×100-CF

b. Sample size of growouts=200-2000 plants from each population(production seed and test plot hybrid seed) at the same environment andsame location.

CASE 2

If the P.P.I. from the hybrid seed production field is similar to theP.P.I. of the test plot hybrid seed, then seed size distributiondifferential between hybrid seed and pollenizer seed or somemorphological traits or molecular markers that can differentiate hybridseed from non-hybrid seed from grow outs need to be used to verify thepercentage of hybridity.

As the invention disclosed herein may be embodied in other specificforms without departing from the spirit or general characteristicsthereof, some of which forms have been indicated, the embodimentsdescribed herein are to be considered in all respects illustrative andnot restrictive. The scope of the invention is to be indicated by theappended claims, rather than by the foregoing description, and allchanges which come within the meaning and range of equivalency of theclaims are intended to be embraced therein.

I claim:
 1. A method for producing a hybrid seed product of abee-pollinated crop, from a cross between female plants from at leastone female line and pollenizer plants from at least one pollenizer line,the hybrid seed product having a percentage of hybridity above apredetermined percentage of hybridity, the method comprising: (a)selecting the at least one female line and the at least one pollenizerline to have a seed coat color differential between seed of the femaleplants and seed of the pollenizer plants; (b) planting the at least onefemale line and the at least one pollenizer line in a production ratioin a production field such that the at least one female line and the atleast one pollenizer lines are crossed to obtain the total seed productcomprising hybrid seed and non-hybrid seed, wherein the hybrid seed andnon-hybrid seed in the production seed each have a seed coat colordistribution and the seed coat color distributions overlap by less thantwenty-five percent of the production seed; and (c) removing at least aportion of the non-hybrid seed from the hybrid seed to achieve apost-production increase of percentage of hybridity and obtain thehybrid seed product having a percentage of hybridity above thepredetermined percentage of hybridity.
 2. The method of claim 1 whereinthe bee-pollinated crop is soybean.
 3. The method of claim 1 wherein thebee-pollinated crop is alfalfa.
 4. The method of claim 1 wherein thepredetermined percentage of hybridity comprises seventy-five percent. 5.The method of claim 1 wherein the production ratio of step (b) isdetermined by selecting a ratio of the female line to the pollenizerline to maximize yield of the total seed product in the production fieldor yield of the hybrid seed product in the production field, whilemaintaining the percentage of hybridity of the hybrid seed product abovethe predetermined percentage of hybridity.
 6. The method of claim 1wherein selecting the at least one female line and the at least onepollenizer line in step (a) comprises: selecting the at least one femaleline and the at least one pollenizer line to provide a cross maintainingat least two-thirds of female seed yield when increasing ratio of femaleplants to pollenizer plants from 1:1 to 3:1 under substantially similarenvironmental conditions.
 7. The method of claim 1 wherein selecting theat least one female line and the at least one pollenizer line comprisesselecting a female line having an average Pollen Production Index(P.P.I.) between 0.0% to 0.42%.
 8. The method of claim 1 whereinselecting the at least one female line and the at least one pollenizerline comprises selecting a pollenizer line having highself-incompatibility.
 9. The method of claim 1 further comprisingcrossing the female plants with the pollenizer plants at a test ratio offemale plants to pollenizer plants in a test plot to obtain a test seedproduct having a test percentage of hybridity, wherein the productionratio of female plants to pollenizer plants needed to produce the hybridseed product having a percentage of hybridity above seventy-five percentis estimated from the test percentage of hybridity.
 10. The method ofclaim 9 wherein the test percentage of hybridity is determined byharvesting the test seed product; and determining the proportion ofhybrid seed in the test seed product.
 11. The method of claim 10 whereincrossing the female plants with the pollenizer plants at the test ratioin the test plot further comprises planting female plants and pollenizerplants in the test plot, the test plot having multiple rows, with thefemale plants planted in a female subrow offset from the centerline ofeach row of the multiple rows on a first side, and the pollenizer plantsplanted in a pollenizer subrow offset from the centerline of each row onan opposite side.
 12. The method of claim 1 wherein substantially allthe non-hybrid seed is removed in step (c).
 13. The method of claim 1further comprising crossing the female plants and the pollenizer plantsat multiple test ratios of female plants to pollenizer plants todetermine a respective test percentage of hybridity for each of themultiple test ratios.
 14. The method of claim 13 wherein the productionratio of step (b) is determined by: performing a regression analysis onthe multiple test ratios test percentage and the respective testpercentage of hybridity; and using the regression analysis to determinethe production ratio of female plants to pollenizer plants to producethe hybrid seed product having a percentage of hybridity aboveseventy-five percent.
 15. The method of claim 1 further comprisingverifying the percentage of hybridity of the hybrid seed product byprogeny testing.
 16. The method of claim 15 wherein verifying thepercentage of hybridity of the hybrid seed product by progeny testingfurther comprises measuring the Pollen Production Index (P.P.I.) of theproduction seed (P.P.I. (Production seed)), measuring the P.P.I. of thetest plot hybrid seed (P.P.I. (Test plot hybrid seed)), and determiningthe percentage of hybridity of the hybrid seed product (% hybridity) as:${\% \mspace{14mu} {Hybridity}} = {\frac{1 - {{P.P.L}\mspace{14mu} \left( {{Production}\mspace{14mu} {seed}} \right)}}{1 - {{P.P.L}\mspace{14mu} \left( {{Test}\mspace{14mu} {plot}\mspace{14mu} {hybrid}\mspace{14mu} {seed}} \right)}} \times 100\%}$17. The method of claim 15 further comprising using molecular markers toidentify the hybrid seed and the non-hybrid seed to determine percentageof hybridity of the hybrid seed product.
 18. The method of claim 15wherein verifying percentage of hybridity for the hybrid seed productfurther comprises: collecting female seed data on traits of the femaleseed; collecting pollenizer seed data on traits of the pollenizer seed;collecting hybrid seed product data on traits of the hybrid seedproduct; and analyzing the female seed data, the pollenizer seed dataand the hybrid seed product data to estimate a percentage of hybridityfor the hybrid seed product.
 19. The method of claim 1 furthercomprising killing the pollenizer plants prior to harvest to increasethe percentage of hybridity of the production seed.
 20. The method ofclaim 1, wherein the portion of non-hybrid seed is removed from thenon-hybrid seed by a color sorter.
 21. The method of claim 1, whereinthe hybrid seed product is produced at commercial levels of production.